Thermophilic bacteria (referred to herein as “thermophiles”) are capable of growth at elevated temperatures. Unlike mesophiles, which grow best at temperatures in the range of 25-40° C., or psychrophilic bacteria, which grow best from 15-20° C., thermophiles grow best at temperatures greater than 50° C. Indeed, some thermophiles grow best at 65-75° C., while hyperthermophiles grow best at temperatures up to 130° C. (Black, Microbiology Principles and Applications, 2d edition, Prentice Hall, New Jersey, 145-146, 1991, herein incorporated by reference).
Thermophiles may be aerobic or anaerobic, and are found in a wide variety of genera and species, including the phototrophic bacteria (e.g., the purple bacteria, green bacteria, and cyanobacteria), eubacteria (e.g., Baccillus, Clostridium, Thiobacillus, Desulfotomaculum, Thermus, lactic acid bacteria, actinomycetes, spirochetes, and numerous other genera), and the archaebacteria (e.g., Pyrococcus, Thermococcus, Thermoplasma, Thermotoga, Sulfolobus, and the methanogens). Accordingly, the environments in which thermophiles are normally found vary greatly, although all of these areas are associated with high temperatures.
Thermophiles, like other bacteria, contain five types of DNA polymerases, termed polymerase I, II, III, IV, and V. Given the nature of thermophile habitats, these enzymes typically exhibit thermostability, and are generally referred to as thermostable DNA polymerases. DNA polymerase I (“Pol I”) is the most abundant polymerase and is generally responsible for certain types of DNA repair, including a repair-like reaction that permits the joining of Okazaki fragments during DNA replication. Pol I is essential for the repair of DNA damage induced by UV irradiation and radiomimetic drugs. DNA polymerase II is thought to play a role in repairing DNA damage that induces the SOS response. In mutants that lack both Pol I and DNA polymerase III, DNA polymerase II repairs UV-induced lesions. DNA polymerase III is a multi-subunit replicase.
Thermostable DNA polymerases have proven very useful in a number of applications in molecular biology. One such application is the polymerase chain reaction (PCR). The PCR process is described, for example, in U.S. Pat. Nos. 4,683,195 and 4,683,202, the disclosures of which are incorporated herein by reference. In a PCR reaction, primers, template, and nucleoside triphosphates are combined in appropriate buffer with a DNA polymerase, for the basic steps of thermal denaturation of target DNA, hybridization of primers to template with cooling of the reaction mixture, and primer extension to produce extension products complementary to template sequences. Thermal denaturation is repeated, primers are annealed to extension products with cooling of the reaction mixture, and previously produced extension products serve as templates for subsequent primer extension reactions. This cycle is repeated a number of times, resulting in an exponential amplification of the desired nucleic acid sequence. Use of a thermostable DNA polymerase provides for repeated heating/cooling cycles without loss of enzyme activity.
A number of applications, for example long range PCR, are hindered by the error rates of Pol I proteins currently available (e.g., Taq DNA Pol I). In addition to decreased error rates, a number of applications would benefit from the use of DNA Pol I exhibiting improved sequence discrimination activity, primer mismatch tolerance, and increased thermostability. For example, a DNA Pol I that tolerates primer mismatches would be useful in PCR methods involving the use of degenerative primers.